Colorectal cancer is the most prevalent cancer in developed countries, accounting for over half a million of deaths annually. Butyrate, a fermentation product of dietary fibre, is a candidate chemotherapeutic agent which specifically induces growth arrest, differentiation and apoptosis of cancer cells in vitro and in vivo. The mitochondria have been shown to play a pivotal role in these butyrate-induced effects, but the precise mechanisms remain unclear. In this study, we investigated mitochondrial proteome changes induced by butyrate in the HCT 116 colon cancer cells, using two-dimensional difference gel electrophoresis. Butyrate altered the expression of 18 well-characterised mitochondrial proteins including metabolic and redox enzymes, respiratory complex subunits, regulators of protein import and translation, and death-inducing factors. One of these proteins, mortalin, was further characterised. Our results showed that a specific mortalin isoform was preferentially down-regulated in the mitochondrial and nuclear fractions of HCT 116, but not in the more resistant HT-29 cell line, upon butyrate-induced apoptosis. This may imply that specific mortalin isoforms are associated with susceptibility to apoptosis.